In vivo involvement of polymorphonuclear neutrophils in Leishmania infantum infection

Abstract

Background: The role of lymphocytes in the specific defence against L. infantum has been well established, but the part played by polynuclear neutrophil (PN) cells in controlling visceral leishmaniasis was much less studied. In this report we examine in vivo the participation of PN in early and late phases of infection by L. infantum.

Results: Promastigote phagocytosis and killing occurs very early after infection, as demonstrated by electron microscopy analyses which show in BALB/c mouse spleen, but not in liver, numerous PN harbouring ultrastructurally degraded parasites. It is shown, using mAb RB6-8C5 directed against mature mouse granulocytes, that in chronically infected mice, long-term PN depletion did not enhance parasite counts neither in liver nor in spleen, indicating that these cells are not involved in the late phase of L. infantum infection. In acute stage of infection, in mouse liver, where L. infantum load is initially larger than that in spleen but resolves spontaneously, there was no significant effect of neutrophils depletion. By contrast, early in infection the neutrophil cells crucially contributed to parasite killing in spleen, since PN depletion, performed before and up to 7 days after the parasite inoculation, resulted in a ten-fold increase of parasite burden.

Conclusions: Taken together these data show that neutrophil cells contribute to the early control of the parasite growth in spleen but not in liver and that these cells have no significant effect late in infection in either of these target organs.

Figure 1

Presence of L. infantum in human PMN (arrow) detected at diagnosis in bone marrow of a VL patient.

Figure 2

Electron microscopy analysis of early parasite phagocytosis in spleen of infected mice. Micrograph of spleen, 1 h after L. infantum inoculation (magnification × 6000).

Figure 3

Neutrophil influx to the infected spleen, shown by electron microscopy. Micrograph of spleen, 24 h after L. infantum inoculation (magnification × 1200)

Figure 4

Electron microscopy analysis of early parasite phagocytosis in liver of infected mice. Micrograph of liver, 1 h after L. infantum inoculation (magnification × 4000).

Figure 5

Influence of RB6-8C5 mAb treatment on CD8+ cell sub-population in spleen, assessed by flow cytometry 22 days after Leishmania and antibody injection, as described in Methods. Results are expressed as percentage of total number of cells in the spleen for each group [mean ± sem (4 mice per group)]. The total number of cells is indicated in the text.

Figure 6

Liver and spleen amastigote load in neutrophil-depleted and control mice. Animals received intraperitoneally 200 μg of RB6-BC5 or of irrelevant IgG2b rat mAb 5 h prior to L. infantum inoculation (10₈ stationary-phase promastigote per mouse). The parasite counts were determined 22 days after the infection from Giemsa-stained spleen and liver touch prints, and are expressed (mean ± SEM) in Leishman Donovan units (LDU = number of amastigotes per 1000 nucleated cells × organ weight (in grams) × 2 × 10₅). The experiment was repeated twice.